The discovery and development of RNA interference and especially the breakthrough of using small interfering RNAs (siRNA) to inhibit gene expression is revolutionizingbiology and opening new avenues for developing novel therapeutics. Despite the current usefulness of the technology, a bottleneck exists for quickly finding effective siRNA target sites in a high throughput-like manner and most researchers currently employ a rational trial-and-error design method. In this proposal, we present a novel cell-based technology to identify effective inhibitors from siRNA libraries (either random or directed). The combination of using a library to find the most effective, robust siRNAs against accessible sites on any given target within a cellular environment, coupled with the inhibitor activity intrinsic to siRNAs, holds great promises in finding effective therapeutics. We have chosen dengue virus, a NIAID Class A threat, as our initial target for developing this technology. Potential target dengue viral sequences will be linked to the HSV thymidine kinase (TK) gene and stably expressed in human cells. Addition of ganciclovir leads to cell killing of any cell expressing the HSV TK protein. Prior to addition of ganciclovir, a proprietary library of siRNAs designed to only target dengue sequences ("directed" library)will be introduced into the cells. Those cells that subsequently survive the killing challenge will be isolated and the rescued siRNAs identified. Among the purified and validated inhibitorsidentified, the most potent anti-dengue siRNAs will be further tested for their anti-dengue viral activity in cell-based and animal models. This approach could potentially be extended to any RNA target of interest, including other viruses and oncogene mRNAs.